1. Field of the Invention (Technical Field)
The present invention relates to the control and treatment of inflammation in mammals, particularly to a method of modulating inflammation with cytochrome P-450 inducers and inhibitors.
2. Background Art
Major advances over the past several years have dramatically enhanced the recognition that inflammation is integral to a broad spectrum of diseases. Inflammation plays a central role in various pulmonary disorders. One of the best examples in this category is asthma. The airway obstruction that defines asthma is associated with inflammation in the lung. Asthma and chronic bronchitis alone may affect 25 million persons in the United States.
Much progress has been made toward understanding the mechanisms of airway inflammation. Information has evolved regarding, among others, airway production of chemokines, cytokines and growth factors, expression of adhesion molecules, and generation of lipid mediators that all orchestrate and control the initiation and propagation of lung inflammation. Based on understanding these pro- and anti-inflammatory mechanisms, new therapeutic approaches are being developed to prevent lung inflammation.
As constituents of endotoxin of the cell walls of gram-negative bacteria, lipopolysaccharides are potent environmental agents that have pro-inflammatory properties and can obstruct and inflame airways. It is known that LPS have a broad range of actions, including the activation of endothelial and epithelial cells, neutrophils, and alveolar macrophages, and the induction of cytokines, prostaglandins, leukotrienes and other inflammatory mediators. Airway infections induced by endotoxin-laden bacteria in hospitalized patients are associated with fever, influx of inflammatory cells (particularly neutrophils), and increased production of respiratory mucus. Previous studies have demonstrated that intra-airway exposure to LPS induces inflammatory changes in the rat and mouse similar to changes in patients with chronic bronchitis, bronchopneumonia, and cystic fibrosis. Hudson A. R., et al., "Granulocyte recruitment to airways exposed to endotoxin aerosols", Am. Rev. Respir. Dis. 115:89-95 (1977); Brigham K. L., et al., "Endotoxin and lung injury", Am. Rev. Respir. Dis. 133:913-927 (1986); Rylander R., et al., "Pulmonary function and symptoms after inhalation of endotoxin", Am. Rev. Respir. Dis. 140:981-996 (1989); Sandstrom T., et al., "Lipopolysaccharide (LPS) inhalation in healthy subjects increases neutrophils, lymphocytes and fibronectin levels in bronchoalveolar lavage fluid", Eur. Respir. J. 5:992-996 (1992); Harkema J. R., et al., "In vivo effects of endotoxin on intraepithelial mucosubstances in rat pulmonary airways. Quantitative histochemistry", Am. J. Pathol. 141:307-317 (1992); Strieter R. M., et al., "Acute lung injury: The role of cytokines in the elicitation of neutrophils", J. Invest. Med. 42:640-651 (1994); Tesfaigzi J., et al., "Induction of EGF receptor and erbB-2 during endotoxin-induced alveolar type II cell proliferation in the rat lung", Int. J. Exp. Path. 77:143-154 (1996).
Arachidonic acid can be oxidized by three enzymatic pathways. McGiff J. C., "Cytochrome P-450 metabolism of arachidonic acid", Annual Rev. Pharmacol. Toxicol. 31:339-369 (1991); Capdevila J. H., et al., "Cytochrome P450 and the metabolism of arachidonic acid and oxygenated eicosanoids", Cytochrome P450: Structure, Mechanism, and Biochemistry. Ortiz de Montellano PR, ed., 2.sup.nd Ed. Plenum Press, New York, pp. 443-471 (1995). Cyclooxygenases (COX) metabolize arachidonic acid to prostaglandins, thromboxane, and prostacyclin. Lipoxygenases (LOX) convert arachidonic acid to leukotrienes and hydroperoxyeicosatetraenoic acids. P-450 monooxygenases catalyze the formation of epoxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, and C19/C20 alcohols of arachidonic acid. Enhanced/altered metabolism of arachidonic acid via COX and LOX is intrinsic to every inflammatory process, including lung inflammation. Henderson W. R., "Eicosanoids and platelet-activating factor in allergic respiratory diseases", Am. Rev. Respir. Dis. 143:S86-S90 (1991); Holtzman M. J., "Arachidonic acid metabolism. Implications of biological chemistry for lung function and disease." Am. Rev. Respir. Dis. 143:188-203 (1991); Shannon V. R., et al., "Histochemical evidence for induction of arachidonate 15-lipoxygenase in airway disease", Am. Rev. Respir. Dis. 147:1024-1028 (1993); Uhlig S., et al., "Cyclooxygenase-2-dependent bronchoconstriction in perfused rat lung exposed to endotoxin", Mol. Med. 2:373-383 (1996).
Pharmacological inhibition of the expression of COX and LOX, as well as modulation of receptors of the respective eicosanoids, play a significant role in modern medicine. Although arachidonic acid can also be converted via P-450 monooxygenase, the significance of this pathway for inflammation has not been thoroughly investigated. However, inflammatory stimuli do affect the expression of various isoforms of cytochrome P-450, mostly in the liver. Morgan E. T., "Regulation of cytochromes P450 during inflammation and infection", Drug Metab. Rev. 29:1129-1188 (1997). Administration of phenobarbital reduces the severity of type II collagen-induced arthritis in the rat. Levy L. E., et al., "Modification of inflammatory process by phenobarbital in rats", Inflammation 15:471-480 (1991). Acetylsalicylic acid, a well known pharmacologic anti-inflammatory inhibitor of cyclooxygenases, has induced P-450 in mouse. Cai Y., et al., "Effect of acetylsalicylic acid on parameters related to peroxisome proliferation in mouse liver", Biochem. Pharmacol. 47:2213-2219 (1994).
As in the other tissues, a high level of COX and LOX has been repeatedly reported in the lung. Holtzman, M. J., "Arachidonic acid metabolism in airway epithelial cells", Annual Rev. Physiol. 54:303-329 (1992). The existence of a third pathway of arachidonic acid oxygenation in the lung--the cytochrome P-450 arachidonate monooxygenase/epoxygenase--was only recently documented. Perdik, P. et al., "Arachidonic acid is metabolized by cytochrome P-450 in the rabbit lung", Am. Rev. Respir. Dis. 147:A920 (1993); Zeldin, D. C., et al., "The rabbit pulmonary cytochrome P-450 arachidonic acid metabolic pathway: characterization and significance", J. Clin. Invest. 95:2150-2160 (1995). However, studies on pulmonary P-450s have a long history, and this enzymatic system plays a significant role in the biotransformation of xenobiotics including inhaled chemical carcinogens and lung toxins. Guengerich, F. P., "Preparation and properties of highly purified cytochrome P450 and NADPH-cytochrome P450 reductase from pulmonary microsomes of untreated rabbits", Mol. Pharmacol. 13:911-923 (1977); Philpot, R. M., et al., "Role of cytochrome P450 and related enzymes in the pulmonary metabolism of xenobiotics", Environ. Health Perspect. 55:359-367 (1984). In contrast to the role for COX and LOX, particularly COX-2 and 15-LOX, no information is available about the role of epoxygenase in lung inflammation.
It is known that interleukin-8 has a role in LPS-induced lung inflammation IL-8 was initially identified as a C--X--C chemokine on the basis of its ability to induce neutrophil activation and chemotaxis. Baggiolini, M., et al, "Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils", J. Clin. Invest. 84:1045-1049 (1989). The recruitment of neutrophils by IL-8, in addition to the action of other chemoattractants on neutrophils, is critical for the propagation of inflammation in the lung. Upon arrival at the lung, activated neutrophils induce pulmonary injury through the release of reactive metabolites and proteolytic enzymes.
Generation of IL-8 is associated with changes in activation of the enzymes involved in arachidonate cascade. Release of IL-8 by human airway smooth muscle cells is stimulated by prostaglandin E.sub.2 (PGE.sub.2) and leukotriene B.sub.4 (LTB.sub.4), and bradykinin-induced production of IL-8 in those cells can be inhibited by COX-2 inhibitors. Interestingly, however, 5,8,11,14-eicosatetraynoic acid (ETYA), an inhibitor of the all three enzymes of the arachidonic acid pathway, and ketoconazole, an inhibitor of LOX and P-450 monooxygenase, increase the induced IL-8 RNA and the release of IL-8 in HL 60 cells. Meier R. W., et al., "Inhibition of the arachidonic acid pathway prevents induction of IL-8 mRNA by phorbol ester and changes the release of IL-8 from HL 60 cells: Differential inhibition of induced expression of IL-8, TNF-alpha, IL-1 alpha, and IL-1 beta", J. Cell Physiol. 165:62-70 (1995).
Enzymes of arachidonate metabolism are also involved in the action of IL-8 on neutrophils. IL-8 induces expression of COX-2 and LOX in human neutrophils, and IL-8-induced activation of neutrophils can be inhibited by the LTB.sub.4 receptor antagonist. Fogh K., et al., "Interleukin-8 stimulates the formation of 15-hydroxy-eicosatetraenoic acid by human neutrophils in vitro", Agents Actions 35:227-231 (1992); Marder P., et al., "Blockade of human neutrophil activation by 2-[2-propyl-3-[2-ethyl-4-(4-fluorophyl)-5-hydroxyphenoxy]propoxy]benzoic acid (LY293111), a novel leukotriene B4 receptor antagonist", Biochem. Pharmacol. 49:1683-1690 (1995).
IL-8 not only promotes inflammation by recruiting neutrophils into the lungs, but also acts to suppress the leukocyte apoptosis. Neutrophil apoptosis is crucial in the resolution of inflammation. One factor that regulates apoptosis is the Bcl-2 gene family, and Bcl-2 extends cell survival under a wide range of stimuli, including infectious and inflammatory conditions. Reed, J. C., "Double identity for proteins of the Bcl-2 family", Nature 387:773-776 (1997); Ibrado A. M., et al., "Bcl-XL overexpression inhibits progression of molecular events leading to paclitaxel-induced apoptosis of human acute myeloid leukemia HL-60 cells", Cancer Res. 57:1109-1115 (1997); Kroemer G., "The proto-oncogene Bcl-2 and its role in regulating apoptosis", Nat. Med. 3:614-620 (1997). Bcl-2 expression is controlled by cytokines. Nagata S., "Fas-mediated apoptosis", Adv. Exp. Med. Biol. 406:119-124 (1996). For example, IL-10 suppresses neutrophil Bcl-2 expression and enhances neutrophil apoptosis, while IL-8 enhances Bcl-2 expression and suppresses neutrophil apoptosis.
Currently, anti-inflammatory drugs belong to two major classes--nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin and ibuprofen, and glucocorticoids. In addition, there are a variety of anti-cytokine drugs in development as well as other drugs that block specific components of inflammation (e.g., anti-adhesion molecules). The NSAIDs are effective against some types of inflammatory processes, and work by inhibiting cyclooxygenase. However, this causes increased production of both lipoxygenase and cytochrome P-450. In some diseases (e.g., asthma), it is thought that the induction of lipoxygenase actually enhances the inflammatory response associated with the disease. Conventional NSAIDs are also problematic in that they are associated with other serious side-effects such as gastrointestinal bleeding and renal dysfunction. Glucocorticoids have many harmful and potentially life-threatening side-effects, including hypertension, hyperglycemia, peptic ulcers, myopathy, cataracts, osteoporosis and osteonecrosis, and growth retardation.
Against the foregoing, the present inventive therapeutic approach was developed. The present invention employs the anti-inflammatory mechanism of cytochrome P-450 inducers of lung inflammation, thereby avoiding some of the drawbacks of current modes of inflammation treatment.